Genome-Driven Investigation of Compatible Solute Biosynthesis Pathways of Pseudomonas syringae pv. syringae and Their Contribution to Water Stress Tolerance

Abstract

The foliar pathogen Pseudomonas syringae pv. syringae exhibits an exceptional ability to survive on asymptomatic plants as an epiphyte. Intermittent wetting events on plants lead to osmotic and matric stresses which must be tolerated for survival as an epiphyte. In this study, we have applied bioinformatic, genetic, and biochemical approaches to address water stress tolerance in P. syringae pv. syringae strain B728a, for which a complete genome sequence is available. P. syringae pv. syringae B728a is able to produce the compatible solutes betaine, ectoine, N -acetylglutaminylglutamine amide (NAGGN), and trehalose. Analysis of osmolyte profiles of P. syringae pv. syringae B728a under a variety of in vitro and in planta conditions reveals that the osmolytes differentially contribute to water stress tolerance in this species and that they interact at the level of transcription to yield a hierarchy of expression. While the interruption of a putative gene cluster coding for NAGGN biosynthesis provided the first experimental evidence of the NAGGN biosynthetic pathway, application of this knockout strain and also a gfp reporter gene fusion strain demonstrated the small contribution of NAGGN to cell survival and desiccation tolerance of P. syringae pv. syringae B728a under in planta conditions. Additionally, detailed investigation of ectC , an orphan of the ectoine cluster (lacking the ectA and ectB homologs), revealed its functionality and that ectoine production could be detected in NaCl-amended cultures of P. syringae pv. syringae B728a to which sterilized leaves of Syringa vulgaris had been added.